POS0331 MOLECULAR MECHANISMS OF CHONDROPROTECTION INDUCED BY ROR2 BLOCKADE
Authors: A. S. Thorup, J. Wilson, M. Deniz, F. Dell’accio
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> We previously reported that ROR2 blockade (ROR2i) induces chondrogenesis <i>in vitro</i> and <i>in vivo</i>, improving pain and cartilage integrity following joint destabilization in mice (Thorup <i>et al.</i>, 2020). Downregulation of YAP signalling was required for chondrogenesis induced by ROR2i, but <i>per se</i> not sufficient. Overexpression of constitutively active YAP negated ROR2i-induced chondrogenesis, but the YAP inhibitor Verteporfin was unable to induce chondrogenesis. This means that something else must take place in addition to YAP inhibition for ROR2i to induce chondrogenesis. <h3>Objectives</h3> In this study, we used a phosphoproteomic approach to detail downstream ROR2 signalling in chondrogenic cells. <h3>Methods</h3> Cartilage differentiation was assessed in C3H10T1/2 cells cultured in micromass using Alcian Blue staining and the expression of mRNA encoding chondrogenic markers. Osteogenesis was assessed by molecular marker analysis, alkaline phosphatase activity and alizarin red staining. Phosphoproteomics screening was performed by precipitation of phosphoproteins on TiO2 columns followed by mass spectroscopy. Ror2-/- C3H10T1/2 cells using CRISPR/Cas9. <h3>Results</h3> In keeping with the delayed endochondral ossification and chondrocyte hypertrophy observed in Ror2-/- mice (DeChiara <i>et al.</i>, 2000), Ror2-/- C3H10T1/2 cells failed to undergo osteogenesis when exposed to osteogenic medium as assessed by alkaline phosphatase activity, extracellular matrix calcification and expression of hypertrophy/osteogenic markers. We established suitable time points for ROR-2 mediated phosphorylation events based on ERK (early) and JNK (late) phosphorylation following stimulation with WNT5A. Phosphoproteomics analysis using a label-free approach, revealed 1109 early (5 min) phosphorylation events that were ROR2 dependent (taking place in the wild type but not in the Ror2-/- CRISPR cells) and 708 at 15 minutes. In keeping with the requirement of YAP for ROR2i-dependent chondrogenesis (Thorup <i>et al.</i>, 2020), several phosphorylation events of molecules within the YAP pathway occurred including YAP and LATS1/2. In keeping with this, YAP dependent transcription was modulated by WNT5A in a ROR2-dependent manner in a reporter assay. Several phosphorylation sites were non-conventional, suggesting a complex, non-canonical, regulation of YAP by ROR2. Other signalling pathways, previously known to be involved in chondrogenesis, were involved including insulin, autophagy and focal adhesion. Many phosphorylation events were previously poorly characterised. <h3>Conclusion</h3> ROR2i induces modulation of several signalling pathway in addition to YAP. The hierarchy of these phosphorylation events needs to be resolved to identify the detailed molecular mechanism underpinning the efficacy of ROR2i in osteoarthritis. <h3>References</h3> [1]DeChiara, T. M. <i>et al.</i> (2000) 'Ror2, encoding a receptor-like tyrosine kinase, is required for cartilage and growth plate development', <i>Nature Genetics</i>. doi: 10.1038/73488. [2]Thorup, A. S. <i>et al.</i> (2020) 'ROR2 blockade as a therapy for osteoarthritis', <i>Science Translational Medicine</i>. doi: 10.1126/SCITRANSLMED.AAX3063. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.